PROJECT SUMMARY/ABSTRACT Due to advances in geroscience, aging must be considered a modifiable risk factor for the major causes of death. Interventions targeting human aging are ongoing, but there is a lack of predictive biomarkers to measure the effect of these candidate interventions. We have identified a mechanism-proximal biomarker that increases exponentially with advancing age ? mitochondrial DNA (mtDNA) deletion mutations. With age, somatically derived mtDNA deletions clonally accumulate within individual cells across a variety of tissues. At high intracellular abundance, mtDNA deletions disrupt oxidative phosphorylation, cellular function, and result in cell death. We have developed a digital PCR assay that quantifies mtDNA deletion frequency using total DNA samples from any tissue in rodents. This assay is absolutely quantitative, amenable to a 96-well format, correlates strongly with the subsequent cellular phenotypes, and has a detection limit below one part per million. In rodents, mtDNA deletion mutation frequency increases exponentially with age and this increase parallels the age-induced accumulation of dysfunctional cells, tissue degeneration, and mortality. This project will develop and validate mtDNA deletion frequency as a biomarker of human aging. We will translate the rodent assay to human mtDNA in accordance with FDA guidelines for bioanalytical assays. We will measure mtDNA deletion frequency in a number of human tissues and biofluids across the human lifespan and establish the relationship between mtDNA deletion frequency and age. Finally, we will test the performance of mtDNA deletion frequency as a predictive biomarker of aging in human longitudinal studies. We will test the hypothesis that mtDNA deletion frequency will predict age-induced morbidity and mortality in humans.